This application is a competing renewal of R01GM066358 for Years 9-13. The project has focused on computational investigations of the anesthetic action on ion channels with the long-term goal of determining the molecular mechanism of general anesthesia. One of the important discoveries from our computational studies of the 1422 and 17 subtypes of nicotinic acetylcholine receptor (nAChRs) is that anesthetic binding to these two nAChR subtypes are similar regardless of their different functional sensitivity to volatile anesthetics. Given that many anesthetic-sensitive and insensitive receptors within the same superfamily share high structure similarity, it is unlikely that anesthetic binding alone can differentiate functional sensitivities to anesthetics. With the recent success in producing ful-length 17 nAChR and its bacterial pentameric channel analogue GLIC, the availability of photoreactive anesthetics and the significant advancement in supercomputing power, this continuation application proposes to test the central hypothesis that only those anesthetic binding sites capable of shifting the equilibriums among the preexisting global conformations of the receptor will have functional impact. The residues forming such binding sites often involve long-range electrostatic interaction. The three new aims are: (1) to produce high quantity and quality full-length 17, 1722, and 1422 nAChRs, as well as GLIC and its anion-conducting mutants, for functional tests of the theoretical predictions. (2) To determine the anesthetic binding sites in the receptors and mutants produced in Aim 1 using photoreactive anesthetic labeling and fluorescence quenching. (3) To reveal underlying mechanisms of anesthetic action on these ion channels through advanced computations, including long-timescale (up to 5s timescale) molecular dynamics simulations to discover where and why only a sub-class of specific anesthetic binding can have functional impact, manifested as either inhibition or potentiation. The innovation of the proposed research is reflected in the novel hypotheses, integrated aproaches, and multidisciplinary collaborations. The significance of the proposed studies is the paradigm-shifting conceptual framework to quantify the action of low-affinity drugs on proteins, providing a new platform for future rational design of novel anesthetics with reduced side effects.